The transcription factor E2F was identified as a sequence-specific DNA binding protein complex required for the adenovirus E1A dependent transcription of the E2 gene, and as a differentiation-regulated transcription factor (also termed DRTF-1) in embryonal carcinoma cells. It is now clear that E2F plays a predominant role in the regulation of genes in G.sub.1 and S. These genes include known regulators of cell proliferation such as c-myc, B-myb, E2F-1 and RB, as well as a number of genes encoding enzymes associated with DNA synthesis, like Dihydrofolate Reductase (DHFR) and Thymidine Kinase (TK). In its simplest transcriptionally active form the transcription factor E2F is a heterodimeric protein composed of one member of the E2F family, which currently consists of 5 distinct gene products (E2F-1 to E2F-5) of which E2F-1 is the factor analysed in greatest detail at present, and one member of the DP family. Three distinct DP genes and their products have been identified, but the most detailed analyses have been performed with DP-1. The heterodimerisation of E2F family members with DP-1 is synergistic, with respect to both DNA binding and E2F-site dependent transactivation. Apart from such dimeric molecules, frequently referred to as "free E2F", higher order complexes exist in the cell, and the formation of the different complexes is dictated by the cell cycle.
In mammalian cells the heterodimeric E2F/DP transcription factors frequently act as repressors in G.sub.0 /early G.sub.1 owing to their association with pocket proteins of the pRb family. In late G.sub.1, the pocket proteins become hyperphosphorylated and dissociate from the complex with E2F/DP, leading to the derepression of E2F-regulated genes. Several genes expressed in late G.sub.1 /early S, including B-myb, DHFR and E2F-1, have been shown to be repressed through an E2F-mediated mechanism in G.sub.0 /G.sub.1 and to be derepressed in late G.sub.1. The E2F molecules include activating dimeric complexes such as, for example, E2F1-DP1 and trimeric E2F complexes such as, for example, E2F1-DP1-pRb Bandara et al., Nature, 252, 249 (1991); Chellappan et al., Cell 65, 1053 (1991); Chittenden et al., Cell 65, 1073 (1991); Helin et al., Cell 70, 337 (1992); Kaelin et al., Cell 70, 351 (1992)!. The complexes E2F4-DP1 and E2F4-DP1-p107 have, for example, been described by Beijersbergen et al., Genes Dev. 8, 2680 (1994) and Ginsberg et al., Genes Dev. 8, 2665 (1994).
A property common to transcription factors, to which the E2F molecules belong, is that there is a relation between the expression of genes and the state in which the cell is. There is very little transcription of genes in quiescent cells, whereas transcription is increased in dividing cells. In an experiment with cultivated cells it is possible to stimulate expression by adding serum, preferably fetal calf serum, to quiescent cells.
Cells multiply by cell division, with the genome of the cell being replicated before mitosis. Non-dividing, quiescent cells are in a phase referred to as G0. Alternatively, non-dividing, quiescent cells are inhibited and cannot progress beyond the G1 stage ("G1 block"). When cell division starts, the cells are released from the G0 phase or the G1 block. The G1 phase is a phase which is normally of relatively long duration. The DNA content of the cell is in the diploid state in the G0 and G1 phases. The G1 phase is followed by the S phase in which DNA synthesis takes place and in which the genome is duplicated. This is followed by the second G phase, G2, in which the cell is in the tetraploid state. Mitosis then takes place, and the cell reaches a G1 or G0 state.
The B-myb gene is involved in cell division. Transcription of the B-myb gene increases (for example in mouse fibroblasts) in the late G1 phase of the cell cycle and is most pronounced in the S phase (Lam et al., EMBO J. 12, 2705 (1993)). The nucleotide sequence CTTGGCGGGAG is a constituent of the promoter sequence of the B-myb gene and is involved in the repression of the gene in the G0 and early G1 phases (Lam et al., EMBO J. 12, 2705 (1993)). TTTCGCGC is a constituent of the E2a adenovirus promoter and represents an activating E2F binding site (Mudryj et al., EMBO J. 9, 2179 (1990)).